A multidrug ABC transporter with a taste for salt.

BACKGROUND: LmrA is a multidrug ATP-binding cassette (ABC) transporter from Lactococcus lactis with no known physiological substrate, which can transport a wide range of chemotherapeutic agents and toxins from the cell. The protein can functionally replace the human homologue ABCB1 (also termed multidrug resistance P-glycoprotein MDR1) in lung fibroblast cells. Even though LmrA mediates ATP-dependent transport, it can use the proton-motive force to transport substrates, such as ethidium bromide, across the membrane by a reversible, H(+)-dependent, secondary-active transport reaction. The mechanism and physiological context of this reaction are not known. METHODOLOGY/PRINCIPAL FINDINGS: We examined ion transport by LmrA in electrophysiological experiments and in transport studies using radioactive ions and fluorescent ion-selective probes. Here we show that LmrA itself can transport NaCl by a similar secondary-active mechanism as observed for ethidium bromide, by mediating apparent H(+)-Na(+)-Cl(-) symport. Remarkably, LmrA activity significantly enhances survival of high-salt adapted lactococcal cells during ionic downshift. CONCLUSIONS/SIGNIFICANCE: The observations on H(+)-Na(+)-Cl(-) co-transport substantiate earlier suggestions of H(+)-coupled transport by LmrA, and indicate a novel link between the activity of LmrA and salt stress. Our findings demonstrate the relevance of investigations into the bioenergetics of substrate translocation by ABC transporters for our understanding of fundamental mechanisms in this superfamily. This study represents the first use of electrophysiological techniques to analyze substrate transport by a purified multidrug transporter.

A super-resolution framework for 3-D high-resolution and high-contrast imaging using 2-D multislice MRI.

A novel super-resolution reconstruction (SRR) framework in magnetic resonance imaging (MRI) is proposed. Its purpose is to produce images of both high resolution and high contrast desirable for image-guided minimally invasive brain surgery. The input data are multiple 2-D multislice inversion recovery MRI scans acquired at orientations with regular angular spacing rotated around a common frequency encoding axis. The output is a 3-D volume of isotropic high resolution. The inversion process resembles a localized projection reconstruction problem. Iterative algorithms for reconstruction are based on the projection onto convex sets (POCS) formalism. Results demonstrate resolution enhancement in simulated phantom studies, and ex vivo and in vivo human brain scans, carried out on clinical scanners. A comparison with previously published SRR methods shows favorable characteristics in the proposed approach.

New structure model for the ATP-binding cassette multidrug transporter LmrA.

Multidrug resistance of pathogenic microorganisms and mammalian tumors can be associated with the overexpression of multidrug transporters. These integral membrane proteins are capable of extruding a wide range of structurally unrelated compounds from the cell. Among the different classes of multidrug transporters are the ATP binding cassette (ABC) transporters, which are dependent on the binding and hydrolysis of ATP. In the past five years, many researchers have built homology models of ABC extrusion systems using the atomic coordinates of crystallized MsbA, a lipopolysaccharide transporter in Gram-negative bacteria. Likewise, we have previously used the Vibrio cholera MsbA structure as a template in the modeling of the multidrug transporter LmrA from Lactococcus lactis. In view of the recently discovered inaccuracies in the MsbA structure, we have remodelled LmrA using the atomic coordinates of the MsbA homologue Sav1866 from Staphylococcus aureus. To compare and test our MsbA-based and Sav1866-based LmrA models we performed cysteine cross-linking at three key positions in LmrA. The pattern of cross-linking at these positions was consistent with the overall topology of transmembrane helices in Sav1866, suggesting that its crystal structure might be physiologically relevant. We recently identified E314 as a residue important in proton conduction by LmrA. The predicted location of this residue at the interface between the two half-transporters in the Sav1866-based homodimer, within the inner leaflet of the phospholipid bilayer, provides a new structural basis for the role of E314 in LmrA-mediated transport.

New light on multidrug binding by an ATP-binding-cassette transporter.

ATP-binding-cassette (ABC) multidrug transporters confer multidrug resistance to pathogenic microorganisms and human tumour cells by mediating the extrusion of structurally unrelated chemotherapeutic drugs from the cell. The molecular basis by which ABC multidrug transporters bind and transport drugs is far from clear. Genetic analyses during the past 14 years reveal that the replacement of many individual amino acids in mammalian multidrug resistance P-glycoproteins can affect cellular resistance to drugs, but these studies have failed to identify specific regions in the primary amino acid sequence that are part of a defined drug-binding pocket. The recent publication of an X-ray crystallographic structure of the bacterial P-glycoprotein homologue MsbA and an MsbA-based homology model of human P-glycoprotein creates an opportunity to compare the original mutagenesis data with the three-dimensional structures of transporters. Our comparisons reveal that mutations that alter specificity are present in three-dimensional 'hotspot' regions in the membrane domains of P-glycoprotein.

Drug-lipid A interactions on the Escherichia coli ABC transporter MsbA.

MsbA is an essential ATP-binding cassette half-transporter in the cytoplasmic membrane of the gram-negative Escherichia coli and is required for the export of lipopolysaccharides (LPS) to the outer membrane, most likely by transporting the lipid A core moiety. Consistent with the homology of MsbA to the multidrug transporter LmrA in the gram-positive Lactococcus lactis, our recent work in E. coli suggested that MsbA might interact with multiple drugs. To enable a more detailed analysis of multidrug transport by MsbA in an environment deficient in LPS, we functionally expressed MsbA in L. lactis. MsbA expression conferred an 86-fold increase in resistance to the macrolide erythromycin. A kinetic characterization of MsbA-mediated ethidium and Hoechst 33342 transport revealed apparent single-site kinetics and competitive inhibition of these transport reactions by vinblastine with K(i) values of 16 and 11 microM, respectively. We also detected a simple noncompetitive inhibition of Hoechst 33342 transport by free lipid A with a K(i) of 57 microM, in a similar range as the K(i) for vinblastine, underscoring the relevance of our LPS-less lactococcal model for studies on MsbA-mediated drug transport. These observations demonstrate the ability of heterologously expressed MsbA to interact with free lipid A and multiple drugs in the absence of auxiliary E. coli proteins. Our transport data provide further functional support for direct LPS-MsbA interactions as observed in a recent crystal structure for MsbA from Salmonella enterica serovar Typhimurium (C. L. Reyes and G. Chang, Science 308:1028-1031, 2005).

A critical role of a carboxylate in proton conduction by the ATP-binding cassette multidrug transporter LmrA.

The ATP binding cassette (ABC) transporter LmrA from the bacterium Lactococcus lactis is a homolog of the human multidrug resistance P-glycoprotein (ABCB1), the activity of which impairs the efficacy of chemotherapy. In a previous study, LmrA was shown to mediate ethidium efflux by an ATP-dependent proton-ethidium symport reaction in which the carboxylate E314 is critical. The functional importance of this key residue for ABC proteins was suggested by its conservation in a wider family of related transporters; however, the structural basis of its role was not apparent. Here, we have used homology modeling to define the structural environment of E314. The residue is nested in a hydrophobic environment that probably elevates its pKa, accounting for the pH dependency of drug efflux that we report in this work. Functional analyses of wild-type and mutant proteins in cells and proteoliposomes support our proposal for the mechanistic role of E314 in proton-coupled ethidium transport. As the carboxylate is known to participate in proton translocation by secondary-active transporters, our observations suggest that this substituent can play a similar role in the activity of ABC transporters.

Reversible transport by the ATP-binding cassette multidrug export pump LmrA: ATP synthesis at the expense of downhill ethidium uptake.

The ATP dependence of ATP-binding cassette (ABC) transporters has led to the widespread acceptance that these systems are unidirectional. Interestingly, in the presence of an inwardly directed ethidium concentration gradient in ATP-depleted cells of Lactococcus lactis, the ABC multidrug transporter LmrA mediated the reverse transport (or uptake) of ethidium with an apparent K(t) of 2.0 microm. This uptake reaction was competitively inhibited by the LmrA substrate vinblastine and was significantly reduced by an E314A substitution in the membrane domain of the transporter. Similar to efflux, LmrA-mediated ethidium uptake was inhibited by the E512Q replacement in the Walker B region of the nucleotide-binding domain of the protein, which strongly reduced its drug-stimulated ATPase activity, consistent with published observations for other ABC transporters. The notion that ethidium uptake is coupled to the catalytic cycle in LmrA was further corroborated by studies in LmrA-containing cells and proteoliposomes in which reverse transport of ethidium was associated with the net synthesis of ATP. Taken together, these data demonstrate that the conformational changes required for drug transport by LmrA are (i) not too far from equilibrium under ATP-depleted conditions to be reversed by appropriate changes in ligand concentrations and (ii) not necessarily coupled to ATP hydrolysis, but associated with a reversible catalytic cycle. These findings and their thermodynamic implications shed new light on the mechanism of energy coupling in ABC transporters and have implications for the development of new modulators that could enable reverse transport-associated drug delivery in cells through their ability to uncouple ATP binding/hydrolysis from multidrug efflux.

An ABC transporter with a secondary-active multidrug translocator domain.

Multidrug resistance, by which cells become resistant to multiple unrelated pharmaceuticals, is due to the extrusion of drugs from the cell's interior by active transporters such as the human multidrug resistance P-glycoprotein. Two major classes of transporters mediate this extrusion. Primary-active transporters are dependent on ATP hydrolysis, whereas secondary-active transporters are driven by electrochemical ion gradients that exist across the plasma membrane. The ATP-binding cassette (ABC) transporter LmrA is a primary drug transporter in Lactococcus lactis that can functionally substitute for P-glycoprotein in lung fibroblast cells. Here we have engineered a truncated LmrA protein that lacks the ATP-binding domain. Surprisingly, this truncated protein mediates a proton-ethidium symport reaction without the requirement for ATP. In other words, it functions as a secondary-active multidrug uptake system. These findings suggest that the evolutionary precursor of LmrA was a secondary-active substrate translocator that acquired an ATP-binding domain to enable primary-active multidrug efflux in L. lactis.

A new dimer interface for an ABC transporter.

The crystallization of MsbA, an ATP-binding cassette (ABC) transporter involved in the transport of Lipid A in Escherichia coli, provided a fascinating glimpse into the high-resolution structure of an ABC transporter at 4.8 A. The E. coli crystal structure of MsbA reveals a dimer. Although the structure of the MsbA monomer is consistent with the biochemistry of ABC transporters, including the human multidrug resistance P-glycoprotein, the interface between the monomers in the MsbA dimer may not reflect the biologically relevant interface. We considered the interface in a two-armed MsbA dimer, named spiral. Our findings indicate that (i) the spiral MsbA dimer may have biological relevance for ABC transporters that interact with lipophilic substrates, and (ii) the dimer interface observed in the crystal structure of E. coli MsbA represents a crystallization artefact. A comparison of the spiral MsbA dimer with the recently published structure of MsbA in Vibrio cholera is also described.